Rheumatoid arthritis (RA) and juvenile rheumatoid arthritis (JRA) are diseases for which current therapies are only partially effective and are associated with significant side effects. Gene therapy delivered locally to diseased synovium is a novel approach offering the potential to achieve steady-state levels of short-lived, specific biologic agents directly to diseased joints, thus minimizing possible toxicity associated with systemic delivery. Recombinant adeno-associated virus (rAAV) vectors have emerged as agents capable of delivering genes to tissues in vivo, including synovium, resulting in very long-term transgene expression. Importantly, a significant and growing number of reports show that rAAV vectors elicit minimal pathogenicity and immunogenicity. Preliminary data from our laboratory demonstrates that both proteasome inhibitors and certain adenovirus proteins dramatically improve rAAV mediated synovial gene transfer. We have also made the novel observation that proteasome inhibitors can regulate transgene expression in AAV-transduced synoviocytes. In this context, the current application will test the hypothesis that rAAV-mediated, localized gene therapy can control human arthritis and will explore the mechanism of proteasome- and adenovirus-enhancement of rAAV-mediated synovial gene transfer. We will determine the specific site or step of transduction at which enhancement by proteasome inhibition and adenovirus helper proteins occur. Using a human RA/JRA-SCID model in which human arthritic synovium is implanted into SCID mice, we will deliver soluble TNF receptor (sTNFR), IL-4 and IL-10 by rAAV-mediated gene transfer. The effects on synovitis and cartilage destruction of local, versus systemic, expression will be compared. To test the effects on synovitis of the removal of sTNFR, IL-4, and IL-10 following a period of expression, we will utilize a system for rAAV-mediated gene transfer under the control of a promoter that is active only in the presence of rapamycin. The potential of proteasome inhibitors to regulate transgene expression will also be determined. rAAV is the first vector with properties sufficiently attractive to be of potential clinical utility in arthritis. The proposed studies will provide a small animal model for testing the potential of rAAV-mediated gene transfer for human arthritides and may serve as the basis for the clinical application of rAAV to the treatment of these disorders.